The elicitation of potent and broad anti-HIV-1 neutralizing antibodies (NAbs) by immunization has been one of the major goals of HIV research since the beginning of the HIV/AIDS epidemic. So far, this goal has not been attained. The target of anti-HIV-1 NAbs is the viral envelope glycoprotein (Env) and several forms of HIV Env have been tested as immunogens over the past three decades. The lack of elicitation of antibodies with broad neutralizing activities by soluble Env immunogens is not due to the absence of relevant epitopes on such constructs. Understanding why conserved neutralization epitopes are poorly immunogenic during immunization by HIV Env constructs is the focus of this proposal. Our overall hypothesis is that the biophysical characteristics of the epitope-BCR interaction, especially the kon and koff binding rates of that interaction, dictate the downstream intracellular events that lead to either B cell-differentiation and antibody production, or to B cell-anergy. To test this hypothesis we propose a series of iterative studies that will improve our understanding of the structural and biophysical characteristics of immunogen-BCR interactions and of how these interactions influence downstream events that lead to B cell activation or anergy. Our team is composed of groups with diverse expertise that have a documented record of collaboration. Dr. Schief's group at the University of Washington is leading our immunogen-design efforts. Dr. Strong's group at the Fred Hutchinson Cancer Research Center is leading our efforts to characterize the structure of immunogens and the antibodies they elicit and their biophysical interactions. Dr. Stamatatos'group is leading our efforts to analyze the interaction of our immunogens with B cells. We are supported in our efforts by Dr. Atchison's group at the Institute for Systems Biology who will define those intracellular events that lead to optimal B cell stimulation and those that lead to B cell anergy or apoptosis, following the binding of b12 scaffolds on the BCR on B cells. PUBLIC HEALTH RELEVANCE (provided by applicant): Our Project focuses on the design of immunogens that would elicit broadly neutralizing antibodies against HIV and in indentifying those intracellular signaling pathways that are required for the production for such antibodies. As such, our studies are highly relevant to the development of an effective vaccine against HIV. PROJECT 1: Title: - Computational design of novel antigens targeting mature and germline b12 Project Leader: SCHIEF, W PROJECT 1 DESCRIPTION (provided by applicant): Here we focus on immunogen design to elicit b12-like antibodies against the conserved cd4 binding site (cd4bs) of HIV Envelope. Many different engineered variants of HIV Envelope have failed to elicit such antibodies, but here we develop two novel types of b12 antigen that have not previously been tested - non- HIV protein scaffolds onto which the b12 epitope has been transplanted, and minimized, stabilized variants of core gp120. Further, we employ a combination of computational protein design and yeast display directed evolution that has not been employed for b12 antigen design previously. We will test the following hypotheses: (i) to induce b12-like antibodies rather than non- or narrowly-neutralizing antibodies against the cd4bs, it will be necessary to design antigens that bind b12 but not other cd4bs antibodies (ii) to optimally stimulate b12 B-cells and elicit b12-like antibodies will require antigens that stabilize the structure of the b12 epitope and optimize the affinity and kinetics of the antigen-b12 interaction (iii) to stimulate naive B cells to develop into those producing the mature broadly-neutralizing form of b12, antigens that bind both germline b12 and mature b12 will be required;(iv) to maximally stimulate b12 B cells by crosslinking B cell receptors, it will be necessary to multimerize b12-antigens on particles in an oriented fashion with the epitope facing outward. Our (Project 1) design efforts will be informed by structural and biophysical analysis of b12 antigens and their interactions with b12 and with mouse MAbs elicited by designed antigens (Project 2), by analysis of b12 antigen stimulation of B cells in vitro and in vivo (Project 3), and by binding specificity and neutralization analysis of rabbit sera elicited by selected b12 antigens (Core B). PUBLIC HEALTH RELEVANCE (provided by applicant): Induction of broadly-neutralizing antibodies (bNAbs) that neutralize diverse strains of HIV will likely be a critical component of a protective HIV vaccine. Here we focus on immunogen design to elicit broadly neutralizing antibodies against the conserved CD4-binding site of the HIV gp120 Envelope protein, using the broadly-neutralizing human monoclonal antibody lgG1b12 as a guide.